PROJECT SUMMARY Ataxia-Telangiectasia (A-T) is a rare (~ 1 in every 100,000) but catastrophic and deadly disease that causes progressive loss of motor function and coordination and death by age 25. In about one-third of cases, the cause is a nonsense mutation in the ATM (Ataxia-Telangiectasia mutated) gene that encodes a premature termination codon (PTC). No effective treatments are available, in part because no satisfactory animal model exists. Atm-/- mice are not a good model because they do not develop ataxia. However, recognizing that the Atm gene product participates in DNA repair pathways, our collaborator Dr. McKinnon reasoned that inflicting a second hit - an additional knockout mutation in a second DNA repair gene that is also linked to ataxia - might produce ataxia. To test this notion, he knocked out the Aptx (aprataxin) gene because: 1) APTX participates in DNA repair; 2) humans lacking APTX protein develop a disease similar to A-T called AOA (Ataxia with Oculomotor Apraxia); and 3) like Atm-/- mice, Aptx-/- mice do not develop ataxia. He crossed the single mutant mice to generate double mutant Atm-/-; Aptx-/- mice that, as anticipated, exhibit the progressive ataxia observed in A-T and AOA patients. Our LONG-TERM GOAL is to: 1) produce a comprehensive understanding of the histopathologic sequence of A-T disease development; 2) explain mechanistically how loss of DNA repair proteins leads to ataxia; and 3) utilize this new mouse model to test new treatments for A-T. AIM 1 proposes hypothesis-generating studies that will dissect the underlying neuropathological and electrophysiological abnormalities that accompany development of ataxia. These experiments, carried out in collaboration with Dr. Jon Cooper (Co-I), will concentrate on the cerebellum and brain regions that project to and from that structure, since it controls motor coordination functions. We will also characterize a new AtmN/N; Aptx-/- genotype that contain a nonsense mutation in the same exon (exon 15) as patients with A-T and that, we predict, will phenocopy Atm-/-; Aptx-/- mice and develop progressive ataxia similar to clinical A-T. AIM 2 will test AtmN/N; Aptx-/- mice and Atm-/-; Aptx-/- controls with our recently developed Small Molecule Read-Through (SMRT) compounds, which efficiently read-through nonsense mutations in the ATM gene (Lee et al., 2013). We will compare our lead candidate (GJ103) with other compounds that can also readthrough nonsense mutations. Dependent variables will include mRNA and protein expression analyses for ATM and downstream molecules, and assessment of motor function using standard tests. We predict that SMRT compounds (but not other readthrough compounds that do not cross the blood-brain barrier) will both restore ATM production in the brain and ameliorate the ataxic phenotype. FUTURE STUDIES: Our results will: 1) provide essential hypothesis-generating preliminary data to fuel mechanistic follow-on studies directed at explaining development of A-T in molecular, cellular, and brain region-specific terms; and 2) provide justification and support for more definitive IND-enabling preclinical development of SMRT compounds to create the first effective treatment for A-T caused by nonsense mutations.